High-dose vitamin C (vitC) has shown superior anticancer activity in immunocompetent compared with immunodeficient mice, suggesting an immunodependent mechanism of action. Because high-dose vitC generates substantial reactive oxygen species (ROS), we hypothesized that ROS could induce immunogenic cell death (ICD), a regulated form of cell death characterized by the release of damage-associated molecular patterns (DAMPs) that enhance antigen presentation and promote antitumor immunity. Spatial transcriptomic and immunofluorescence analyses were performed on breast and colon tumors from mice treated with high-dose vitC to quantify oxidative stress, DAMP mobilization, and immune cell infiltration. To dissect the functional role of ROS and ICD, mice were treated with high-dose vitC in combination with the antioxidant N-acetylcysteine or an antibody blocking calreticulin (an early DAMP essential for ICD induction). VitC treatment caused marked oxidative stress in tumors from both immunocompetent and immunocompromised mice; however, tumor growth inhibition occurred exclusively in immunocompetent animals. High-dose vitC triggered ROS-dependent exposure and release of the DAMPs calreticulin and HMGB1, respectively. This was accompanied by substantial remodeling of the tumor microenvironment, including increased infiltration of cytotoxic CD8⁺ T cells and natural killer cells, and a reduction in immunosuppressive regulatory T cells. Blocking calreticulin effectively disrupted the ICD cascade, prevented immune microenvironment remodeling, and completely abrogated the antitumor efficacy of vitC. Collectively, our study indicates that high-dose vitC promotes ICD by mobilizing calreticulin and HMGB1, and that calreticulin is a determinant of its immune-mediated antitumor activity. Implications: vitC induces ICD, supporting its use as an adjuvant to anticancer immunotherapy.
Abstract Radioresistance remains a major barrier to effective breast cancer treatment, yet the molecular programs underlying acquired resistance remain incompletely understood. By establishing and characterizing radioresistant breast cancer cell models across multiple molecular subtypes, we revealed enhanced DNA repair capacity and epithelial-mesenchymal transition features in resistant cells. Transcriptomic profiling and functional validation identified platelet-derived growth factor B (PDGFB) as a key regulator of radioresistance. High PDGFB expression was associated with adverse outcomes across independent breast cancer cohorts, including a radiotherapy-treated subgroup. Mechanistically, PDGFB associated with DEAD-box RNA helicase 5 (DDX5) and stabilized DDX5 by limiting its ubiquitin-proteasome-mediated degradation. PDGFB depletion impaired homologous recombination (HR) efficiency, delayed DNA damage resolution, reduced RAD51 foci formation, and resensitized radioresistant cells to irradiation in vitro and in vivo. Conversely, DDX5 overexpression partially restored HR signaling, DNA damage resolution, and clonogenic survival in PDGFB-depleted cells. These findings uncovered a PDGFB-DDX5 regulatory axis that preferentially targets homologous recombination-associated DNA repair and contributes to breast cancer radioresistance. Implications: This study demonstrates the PDGFB–DDX5 axis as a crucial mechanism contributing to radioresistance by preferentially affects HR–mediated DNA damage repair, which can be therapeutically explored for overcoming radioresistance in breast cancer.
Abstract Bladder cancer is a major malignancy worldwide. Although cigarette smoking is the leading modifiable risk factor, the contribution of waterpipe smoking (WPS), which is increasingly popular globally and often misperceived as safer, remains unclear. In 2019, Lebanese males had the highest bladder cancer incidence in the Arab world (five times the global average), prompting investigation of WPS as a potential carcinogen. We performed a cross-sectional genomic analysis of patients with exclusive WPS and urothelial carcinoma treated at the American University of Beirut Medical Center (n=250 screened; 14 exclusively WPS), integrating targeted sequencing (MSK-IMPACT®) and whole-exome sequencing (TEMPO pipeline). Tumor mutational burden (TMB), microsatellite instability, mutational signatures, and oncogenic alterations were assessed. Previously profiled bladder cancer tumors from cigarette smokers and non-smokers at Memorial Sloan Kettering Cancer Center (n=751) served as comparators. MSK-IMPACT® successfully profiled 13 tumors. WPS-associated tumors exhibited a numerically higher median TMB than cigarette (20 vs. 10 mut/Mb; p=0.10, q=0.2), with 38.5% classified as MSS-hypermutated, a phenotype associated with elevated mutational load despite microsatellite stability. Common mutations included TERT promoter (62%), TP53 and FGFR3 (46%), and PIK3CA (38%). APOBEC mutational signatures predominated across all cohorts, suggesting shared mutagenic processes, while chromatin-modifying gene alterations were less frequent. PD-L1 expression was generally low. Implications: Exclusive WPS induces bladder tumors with overall genomic features resembling cigarette-associated disease, yet with numerically higher TMB and enrichment of MSS-hypermutated phenotypes. These findings provide novel mechanistic evidence of tobacco-related carcinogenesis beyond cigarettes and highlight potential biomarker relevance for immunotherapy in a globally rising exposure group.
Abstract Transcription factor fusions are recurrent drivers of sarcomas and other cancers, but their oncogenic reorganization of chromatin is difficult to target. One such fusion, PAX3::FOXO1, drives an aggressive subset of pediatric rhabdomyosarcoma (RMS) through chromatin remodeling, blocking terminal differentiation and sustaining proliferation. We hypothesized that identifying PAX3::FOXO1 genetic dependencies would reveal new facets of transcription factor fusion biology. We report an unexpected role for PAX3::FOXO1 in driving DNA replication fork progression, uncovered by its dependence on the INO80 chromatin remodeling complex (INO80c). Loss of the INO80c member UCHL5 reduces the competitive fitness of fusion positive (FP), but not fusion negative (FN) RMS cell lines, and is accompanied by reduced DNA replication fork speed. UCHL5 is required to maintain the N-terminal subdomain of INO80c, but a catalytically dead UCHL5 is sufficient to rescue complex formation, fork progression, and FP-RMS fitness. Similarly, loss of PAX3::FOXO1 rapidly leads to RPA32 foci and slows DNA fork progression. These findings demonstrate that PAX3::FOXO1 and INO80c are each critical to maintain replication fork progression in FP-RMS, a novel function for this transcription factor fusion. Implications: PAX3::FOXO1 and the N-terminal INO80 complex are both required to drive replication fork progression in FP-RMS, offering a counterpoint to models in which fusion oncogenes drive replication stress, and identifying INO80 complex members as a genetic dependency in FP-RMS cells.
Abstract Cancer-related somatic histone mutants are present in 4% of diverse tumor types and display a similar genomic prevalence to other somatic mutations in cancer, but they are not well characterized. We curated and characterized 17 of the most frequently recurring missense mutations in histone H2A, H2B, and H3 genes in breast cancers occurring at glutamate (E), aspartate (D), serine (S), and arginine (R) residues. Many of these mutants co-occur with PIK3CA mutations in breast cancers. We expressed these breast cancer-associated histone mutants in MCF-7 and MDA-MB-468 breast cancer cells, or MCF-10A mammary epithelial cells, and screened them for effects on oncogenic phenotypes in a variety of cell- and tumor-based assays (i.e., proliferation, migration, invasion, competitive outgrowth, transformation). In addition, we examined the effects of selected mutants on DNA damage, gene expression, and histone modifications. We observed varying phenotypic and functional effects. Some of the histone mutants promoted cancer-related phenotypes, with H2B-E76Q, H3-E97K, and H3-E105K eliciting strong oncogenic phenotypes, alterations in gene expression, and context-dependent growth enhancement in a PI3K-activated background. We further showed that the H2B-E76Q and H3-E105K mutants exhibit reduced ADP-ribosylation compared to their cognate wild-type histones, resulting in altered H3-S10 phosphorylation and H3-K27 acetylation in cells. Our results indicate that some histone mutants can promote oncogenic effects in breast cancers, contributing to cancer pathogenesis and tumor progression. Implications: These findings identify a subset of breast cancer-associated histone variants with oncogenic activity, highlighting the role of histone mutations in promoting tumor phenotypes through epigenomic alterations and signaling crosstalk.
